US11464850B2 - Recombinant RSV antigens, nucleic acid molecules encoding the antigens, and vaccine compositions comprising the same - Google Patents
Recombinant RSV antigens, nucleic acid molecules encoding the antigens, and vaccine compositions comprising the same Download PDFInfo
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- US11464850B2 US11464850B2 US16/462,757 US201716462757A US11464850B2 US 11464850 B2 US11464850 B2 US 11464850B2 US 201716462757 A US201716462757 A US 201716462757A US 11464850 B2 US11464850 B2 US 11464850B2
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Definitions
- the present disclosure relates to antigens, nucleic acid molecules, and vaccine compositions for elicitation of an immune response, and more particularly for eliciting an immune response against a respiratory syncytial virus (RSV).
- RSV respiratory syncytial virus
- RSV has been recognized as the most common cause of lower respiratory tract infections in infants and young children. According to the report from World Health Organization, RSV is responsible for an estimated 199,000 deaths annually worldwide, and 99% of which occur in developing countries (Nair, H., et al., 2011). RSV can also infect and cause diseases in people of all ages, most severely in the elderly and in immuno-compromised individuals. Most children are infected at least once by age 2 and continue to be re-infected throughout life possibly due to incomplete immunity to RSV (Hall, C. B., et al., 1991).
- a humanized monoclonal antibody comprises 95% human antibody sequence and 5% murine antibody sequences.
- Palivizumab can treat lower respiratory tract disease caused by RSV by binding to an epitope in the A antigenic site of the F subunit of RSV.
- palivizumab is licensed only for use in high-risk infants.
- the antiviral agent, ribavirin has a therapeutic effect on RSV pneumonia and bronchiolitis, while ribavirin is used to treat RSV infection only in the pediatric population.
- ribavirin is accompanied by many side effects, e.g., insomnia, dyspnea, headache, nausea, muscle pains, emotional lability, hemolytic anemia, decreased hemoglobin, allergic reactions and liver problems. Moreover, ribavirin is not recommended for pregnant women due to its potential risk to the baby.
- FIRSV formalin-inactivated RSV
- VLPs virus like particles
- subunits subunits
- live-attenuated vaccines there is still no licensed vaccine against RSV infections. Due to a tremendous disease burden and limited prophylactic method, the demand for a safe and effective RSV vaccine is now greater than ever.
- the present disclosure provides an antigen comprising a recombinant respiratory syncytial virus (RSV) F protein to mimic the natural trimeric conformation of the RSV F protein.
- RSV respiratory syncytial virus
- the recombinant RSV F protein comprises an antigenic region flanked with an N-terminal heptad repeat (HRN) region and a C-terminal heptad repeat (HRC) region, wherein the antigenic region comprises one or more antigenic sites selected from the group consisting of site ⁇ , site II, and site IV, provided that if the antigenic region comprises more than one antigenic sites, then the antigenic sites are linked to each other by a linker, and the linker, on each occurrence, independently consists of 2 to 20 amino acids.
- HRN N-terminal heptad repeat
- HRC C-terminal heptad repeat
- the antigenic region comprises site ⁇ , site II, and site IV.
- the HRN region is directly linked to site ⁇ , and the HRC region is linked to site IV by the linker.
- the present disclosure provides a nucleic acid molecule encoding the recombinant RSV F antigen described above.
- the present disclosure provides a vaccine composition comprising an effective amount of the antigen or the nucleic acid molecule described above.
- the vaccine composition comprising an effective amount of one or more of the antigens described above and a nucleic acid molecule or a plasmid encoding or expressing the antigen is administered to a subject in need thereof under a condition sufficient to prevent or ameliorate an RSV infection in the subject.
- the vaccine composition is administered in an amount sufficient to elicit an immune response against an RSV antigen, such as RSV F protein, in the subject.
- the present disclosure provides a recombinant RSV F protein and a nucleic acid molecule encoding the recombinant RSV F protein. Also, the present disclosure provides a vaccine composition comprising an effective amount of the antigen or the nucleic acid molecule.
- the antigen, nucleic acid molecule and vaccine composition of the present disclosure can induce antibody responses specific for RSV and protect the subject from RSV infection without causing an adverse effect. Further, compared with the wild-type RSV F protein, the recombinant RSV F protein of the present disclosure is shorter in length and can result in a better expression level. As such, the antigen, nucleic acid molecule and vaccine composition of the present disclosure are relatively easy in mass production and more helpful in increasing the specificity of antibody identification and avoiding unnecessary reactions such as allergy.
- FIGS. 1A-1C show the schematic diagrams of HR ⁇ 24, HR ⁇ , and HR ⁇ -3 ⁇ recombinant proteins, respectively.
- FIGS. 2A-2I are results of SDS-PAGE analysis of HR ⁇ 24, HR ⁇ , HR ⁇ -3 ⁇ recombinant proteins, and HBc.
- FIGS. 2A, 2C, 2E, and 2G show Coomassie blue staining of purified HR ⁇ 24, HR ⁇ , HR ⁇ -3 ⁇ recombinant proteins, and HBc, respectively;
- FIGS. 2B, 2D, and 2F show Western blotting of purified HR ⁇ 24, HR ⁇ , and HR ⁇ -3 ⁇ recombinant proteins using anti-His antibody, respectively;
- FIG. 2H shows Western blotting of purified HBc using rabbit polyclonal anti-HBc antibody; and
- FIG. 2I shows Western blotting of purified HBc and HR ⁇ 24 recombinant proteins using mouse monoclonal anti-RSV antibody, respectively.
- FIGS. 3A and 3B show the transmission electron microscope (TEM) images of purified HBc and HR ⁇ 24 recombinant proteins, respectively.
- FIG. 4 shows the intranasal (IN) immunization schedule.
- Groups of mice are immunized 3 times with vaccine candidates on week 0, 3, and 6, and received RSV challenge on week 9.
- a group immunized with formalin-fixed RSV (FIRSV) intramuscularly (i.m) on week 4 before RSV challenge is also included.
- FIRSV formalin-fixed RSV
- BALF Mouse serum and bronchoalveolar lavage fluid
- FIGS. 5A-5E show HR ⁇ 24-specific antibody responses in mice received 3 doses of intranasal administration of HR ⁇ 24, HR ⁇ , or HR ⁇ -3 ⁇ mixed with or without HBc or CpG. Serum and BALF are collected from the mice 2 days before RSV challenge.
- FIGS. 5A, 5B, 5C , and 5 E show HR ⁇ 24-specific total IgG, IgG1, IgG2a and IgA responses measured from the serum, respectively; and
- FIG. 5D shows HR ⁇ 24-specific secretary IgA (sIgA) response detected from the BALF.
- FIGS. 6A and 6B show mouse body weight changes after RSV challenge.
- the body weight of naive or vaccinated mice is monitored for 5 days after RSV challenge.
- Body weight changes are presented as the weight loss percentage compared to day 0.
- FIG. 7 shows lung histopathology. Lung tissues are collected from naive or vaccinated mice at day 5 post RSV challenge for histology analysis.
- FIG. 8 shows lung virus load. Lung tissues are collected from naive or vaccinated mice at day 5 post challenge for lung virus load analysis by qRT-PCR targeting RSV N gene.
- an antigen includes mixtures of antigens
- a pharmaceutically acceptable carrier includes mixtures of two or more such carriers, and the like.
- the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
- the present disclosure provides an antigen comprising a recombinant RSV F protein.
- the recombinant RSV F protein comprises an antigenic region flanked with an HRN region and an HRC region, and the antigenic region comprises one or more antigenic sites selected from the group consisting of site ⁇ , site II, and site IV.
- RSV has three surface glycoproteins, i.e., small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F).
- SH small hydrophobic
- G attachment
- F fusion
- NT neutralizing
- 5C4 shares these properties with two other antibodies isolated from immortalized peripheral blood mononuclear cells (PBMCs), D25 and AM22, which have been shown to neutralize RSV with 100 folds greater potency than palivizumab (McLellan, J. S., et al., 2013).
- D25 and AM22 target site ⁇ , a metastable antigenic site located on the surface of the prefusion RSV F trimer (Spits, H., et al., 2010; Beaumont, T., et al., 2012).
- the prefusion and postfusion crystal structures of F protein suggest that while sites II and IV are found on both structures, site ⁇ appear to be specific for the prefusion form (McLellan, J. S., et al., 2013).
- the fusion peptide region of RSV F is located at the N terminus of the F1 subunit (Collins, P. L., et al., 1996) while the transmembrane segment contains two regions of 4,3-hydrophobic heptad repeats (HR), a sequence motif suggestive of coiled-coil structures (Chambers, P., et al., 1990; Singh, M., et al., 1999). These regions are denoted as HRN and HRC, respectively, and are separated by an intervening domain of about 270 amino acids. HRN and HRC form a trimeric hairpin-like structure, with the HRC regions packing in an antiparallel manner against the inner coiled-coil formed by HRN regions (Baker, K. A., et al., 1999).
- the HRN region and the HRC region comprise amino acid sequences at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NO: 1 (MAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNK QS) and SEQ ID NO: 2 (NFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK) and have the same functions as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
- the antigenic site comprised in the antigenic region may be selected from the group consisting of site ⁇ , site II, and site IV.
- the site ⁇ , site II, and site IV comprise amino acid sequences at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NO: 3 (KNYIDKQLLPIVNK), SEQ ID NO: 4 (NSELLSLINDMPITNDQKKLMSN), and SEQ ID NO: 5 (KNRGIIKTFS) and have the same functions as SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.
- the antigenic region comprises more than one antigenic sites, then the antigenic sites are linked to each other by a linker, and the linker, on each occurrence, independently consists of 2 to 20 amino acids.
- sequence identity or, for example, comprising a “sequence 80% identical to” refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
- a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
- the identical nucleic acid base e.g., A, T, C, G, U
- the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys
- nucleotides and polypeptides having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity or function of the reference polypeptide.
- the antigenic region comprises at least one site ⁇ directly linked to the HRN region.
- the antigenic region comprises one site ⁇ directly linked to the HRN region and linked to the HRC region by the linker (see, e.g., FIG. 1 ).
- the recombinant RSV F protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 (MAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNK QSGSGSGSEFGGSGNFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKLE HIEHHHH) and has the same function as SEQ ID NO: 6.
- the antigenic region comprises four site ⁇ , and one of which is directly linked to the HRN region (see, e.g., FIG. 1C ).
- the recombinant RSV F protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7 (MAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNK QSGSGSASSNIKENKCNAAKNYIDKQLLPIVNKGGGSSNIKENKCNAAKNYIDKQLLPIV NKGGEFSNIKENKCNAAKNYIDKQLLPIVNKKLGGSGNFYDPLVFPSDEFDASISQVNEK INQSLAFIRKSDELLHNVNAGKLE) and has the same function as SEQ ID NO: 7.
- the antigenic region comprises site ⁇ , site II, and site IV.
- the HRN region is directly linked to site ⁇ , and the HRC region is linked to site IV by the linker (see, e.g., FIG. 1A ).
- the recombinant RSV F protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8 (MAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNK QSGSGSNSELLSLINDMPITNDQKKLMSNNVQIVRQGGGSCTASNKNRGIIKTFSNGGGS GNFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK) and has the same function as SEQ ID NO: 8.
- the linker on each occurrence, independently consists of 2 to 20 amino acids, and the linker may independently include, but not limited to, an amino acid sequence of any one of GSGS (SEQ ID NO: 38), GGGS (SEQ ID NO: 39), GGSG (SEQ ID NO: 40), SGSG (SEQ ID NO: 41) and GG (SEQ ID NO: 42).
- the antigen specifically binds to a 5C4, a D25, or an AM22 prefusion-specific antibody.
- the present disclosure provides a nucleic acid molecule encoding the antigen described above.
- the nucleic acid molecule is codon optimized for expression in a prokaryotic cell.
- the prokaryotic cell is an Escherichia coli cell.
- the nucleic acid molecule comprises a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 9.
- the nucleic acid molecule is codon optimized for expression in a eukaryotic cell.
- the eukaryotic cell is a yeast cell or a mammalian cell.
- the mammalian cell is a human cell.
- the present disclosure provides a vaccine composition comprising an effective amount of the antigen or the nucleic acid molecule described above.
- the vaccine composition may be used to induce an immune response to RSV in a subject.
- a therapeutically effective amount of a vaccine composition comprising one or more of the antigens of the present disclosure, or a nucleic acid molecule or a plasmid encoding or expressing the antigen, can be administered to a subject to elicit an immune response to RSV.
- a therapeutically effective amount of a vaccine composition comprising one or more of the antigens of the present disclosure, or a nucleic acid molecule or a plasmid encoding or expressing the antigen, is administered to a subject in need under conditions sufficient to prevent or ameliorate an RSV infection in the subject.
- the vaccine composition is administered in an amount sufficient to elicit an immune response against an RSV antigen, such as RSV F protein, in the subject.
- the vaccine composition of the present disclosure may be administered intranasally or intramuscularly to the subject.
- the vaccine composition may further comprise a pharmaceutically acceptable carrier and/or an adjuvant.
- the “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like which may be appropriate for administration of the vaccine composition of the present disclosure.
- the pharmaceutically acceptable carrier useful for the present disclosure may include, but not limited to, a preservative, a suspending agent, a tackifier, an isotonicity agent, a buffering agent, and a humectant.
- the adjuvant useful for the present disclosure may include, but not limited to, a CpG oligonucleotide and a hepatitis B core virus-like particle (HBc VLP).
- the vaccine composition administered to the subject comprises a mixture of the antigen and the adjuvant at a weight ratio of 10:1 to 1:10.
- the vaccine composition promotes a Thl immune response.
- RSV F protein Full length cDNA sequence of RSV F protein with optimized codon for Escherichia coli ( E. coli ) expression was synthesized (Genomics BioSci & Tech). Using this sequence as the PCR template, four gene fragments of RSV F protein were amplified, including nucleotides 457-633 which contain HRN and site ⁇ (SEQ ID NO: 10), nucleotides 760-849 which contain site II (SEQ ID NO: 11), nucleotides 1264-1314 which contain site IV (SEQ ID NO: 12), and nucleotides 1426-1560 which contain the C-terminal ⁇ -helix (HRC) (SEQ ID NO: 13).
- PCR amplicons were linked by overlapping PCR and connected by a glycine-rich linker, such as GSGS (SEQ ID NO: 38), GGGS (SEQ ID NO: 39), GGSG (SEQ ID NO: 40), SGSG (SEQ ID NO: 41) and GG (SEQ ID NO: 42), to form a constructed gene (named HR ⁇ 24), which was then inserted into the NcoI-XhoI restriction sites of pET28b tagged with 6-His at the C-terminus to obtain an HR ⁇ 24 plasmid.
- a glycine-rich linker such as GSGS (SEQ ID NO: 38), GGGS (SEQ ID NO: 39), GGSG (SEQ ID NO: 40), SGSG (SEQ ID NO: 41) and GG (SEQ ID NO: 42)
- HR ⁇ plasmids For the construction of HR ⁇ plasmids, two gene fragments of RSV F protein represented by SEQ ID NOs: 10 and 13 were amplified. These two PCR amplicons were then inserted into the NcoI/BamHI and EcoRI/XhoI restriction sites of pET28a tagged with 6-His at the C-terminus and connected by a glycine-rich linker to obtain an HR ⁇ plasmid.
- HR ⁇ -3 ⁇ plasmids For the construction of HR ⁇ -3 ⁇ plasmids, two gene fragments of RSV F protein represented by SEQ ID NOs: 10 and 13 were amplified. Further, three site 0 fragments containing NheI/BamHI, BamHI/EcoRI, or EcoRI/HindIII restriction sites were created by PCR. These five PCR amplicons were then inserted into the NcoI/NheI/BamHI/EcoRI/HindIII/XhoI restriction sites of pET28a tagged with 6-His at the C-terminus and connected by a glycine-rich linker to obtain an HR ⁇ -3 ⁇ plasmid.
- the resulting plasmids were transformed into E. coli BL21 (DE3) competent cells for protein expression.
- the schematic diagrams of HR ⁇ 24, HR ⁇ , and HR ⁇ -3 ⁇ recombinant proteins were shown in FIGS. 1A-1C , respectively.
- the recombinant RSV F protein-6-His and HBc-6-His were expressed in the transformed E. coli BL21 (DE3) obtained from Examples 1 and 2, and purified using nickel affinity chromatography, respectively. Eluted (with 500 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0) protein was buffer exchanged by gradient dialyzing 1 volume of sample against 200 volumes of dialyzing buffer (from 350 mM, 150 mM to 0 mM imidazole in 1 ⁇ PBS) for 12 h in each step. The dialyzed protein-6-His was concentrated using a centrifugal concentrator (10,000 MWCO, Sartorius) to reach a concentration about 1 mg/mL. Molecule size and purity of the protein were determined by SDS-PAGE.
- FIGS. 2B, 2D, and 2F A band of identical mobility was detected by immunoblotting using antibodies directed against His tag, and the results were shown in FIGS. 2B, 2D, and 2F .
- Densitometric scanning of Coomassie blue stained gels revealed that the purified proteins HR ⁇ 24, HR ⁇ , HR ⁇ -3 ⁇ and HBc amounted to more than 90% of the total protein ( FIGS. 2A, 2C, 2E, and 2G ), which was sufficiently pure for immunizations.
- the HBc VLPs have been confirmed to form virus-like particles by TEM ( FIG. 3A ).
- the TEM image also showed that the recombinant HR ⁇ 24 protein formed about 50 nm polymerized nanoparticles ( FIG. 3B ).
- RSV A2 strain was obtained from ATCC. Propagation of the virus was performed in HEp-2 cells ATCC. Cells grown in 100 mm Petri dish (Thermo Scientific) up to 80% confluency were inoculated with RSV A2 at an m.o.i. (multiplicity of infection) of 0.2. Virus adsorption was carried out in serum free Dulbecco's Modified Eagle's medium (DMEM) in a CO 2 incubator at 37° C. After 2 hours, medium was replaced with DMEM supplemented with 2% fetal bovine serum, and the dishes were incubated for another 48 to 72 hours. Supernatants which contain the virus were separated from cell debris by centrifugation at 3,000 rpm for 10 min. Virus was then concentrated by a centrifugal concentrator (100,000 MWCO, Sartorius).
- DMEM serum free Dulbecco's Modified Eagle's medium
- RSV virus titer was determined by plaque assay. Confluent monolayer of HEp-2 cells in 12-well plates were washed with 1 ⁇ PB S and then infected with RSV A2 virus at various dilutions (10 ⁇ 3 to 10 ⁇ 7 ). After 2 hours of virus adsorption, supernatant was removed, and the cell monolayer was washed with 1 ⁇ PBS, followed by overlaying with DMEM+2% fetal bovine serum+0.3% agarose. After 5 days incubation at 37° C. in a CO 2 incubator, cells were fixed with 10% formalin and stained with 0.05% crystal violet for plaque quantification.
- Pathogen-free C57BL/6J female mice (6-8 weeks old) were randomly divided into several groups and immunized by the intranasal (i.n) route with vaccine candidates on day 0, 21, and 42 and challenged with 1 ⁇ 10 6 p.f.u. RSV on day 63 ( FIG. 4 ).
- the vaccine candidates include: 50 ⁇ g HR ⁇ 24; 25 ⁇ g HBc VLPs+25 ⁇ g HR ⁇ 24; 25 ⁇ g HBc VLPs+25 ⁇ g HR ⁇ 24+20 ⁇ g CpG (TCGTCGTTTTCGGCGCGCGCGCCG, SEQ ID NO.
- a naive control group and a group immunized with 25 ⁇ g HBc VLPs intranasally on day 0, 21, and 42 were included.
- a group immunized with 1 ⁇ 10 5 p.f.u. FIRSV intramuscularly (i.m) on day 35 was also included.
- mice serum and bronchoalveolar lavage fluid were collected from separate groups with identical dosing regimen on day 61.
- BALF bronchoalveolar lavage fluid
- the mice were anesthetized with 1.5% isoflurane and then infected by intranasal inoculation of 1 ⁇ 10 6 p.f.u. RSV on day 63.
- body weights of the mice were monitored for 5 days.
- the mice were sacrificed on day 68, and the individual lungs were collected for virus load and histopathology experiments.
- Serum and BALF collected from the immunized mice as described in Example 5 were tested for antibody responses by enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well plate was coated with 50 ⁇ L of purified HR ⁇ 24 (10 ⁇ g/ml) overnight at 4° C. The plate was blocked with 2% BSA for 1 hour at 37° C., and incubated with serial dilutions of serum samples (10 ⁇ 2 to 5.12 ⁇ 10 ⁇ 4 ) or BALF (10 ⁇ 1 to 1.28 ⁇ 10 ⁇ 3 ) in assay diluent (1% BSA, 0.05% Tween 20 in 1 ⁇ PBS) for 2 hours at room temperature.
- assay diluent 1% BSA, 0.05% Tween 20 in 1 ⁇ PBS
- Dilution curve was drawn for each sample, and endpoint titers were calculated as the reciprocal of the dilution producing an optical density that was 0.1 U greater than the background value (1/50 dilution of a pooled pre-immune serum or 1/5 dilution of a pooled naive BALF).
- IgG titers lower than 50 (negative samples) or secretory IgA (sIgA) titers lower than 5 were arbitrarily assigned as 50 or 5.
- HR ⁇ 24, HR ⁇ and HR ⁇ -3 ⁇ can elicit serum HR ⁇ 24-specific total IgG, IgG1, IgG2a, and lung HR ⁇ 24-specific sIgA. Furthermore, by using HBc as an adjuvant, HR ⁇ 24, HR ⁇ and HR ⁇ -3 ⁇ can elicit significant higher serum HR ⁇ 24-specific total IgG, IgG1, IgG2a, IgA and lung HR ⁇ 24-specific sIgA, in which the highest end-point titers were observed in the HBc/HR ⁇ 24 group.
- HR ⁇ 24 can also elicit higher serum HR ⁇ 24-specific total IgG, IgG1, IgG2a, IgA and lung HR ⁇ 24-specific sIgA.
- mice immunized with HR ⁇ 24/HBc, HR ⁇ 24/HBc/CpG, HR ⁇ -30, HR ⁇ -30/HBc, HR ⁇ -30/HBc/CpG, HR ⁇ /HBc or HR ⁇ /HBc/CpG showed less body weight loss compared with the naive group after day 2 post challenge. More specifically, FIG. 6A showed about 8-11% body weight loss in the groups of mice received HR ⁇ 24 mixed with HBc.
- mice immunized with 25 ⁇ g and 50 ⁇ g HR ⁇ 24 mixed with HBc/CpG showed about 16% and 12% body weight loss, respectively, at day 3 post challenge.
- FIG. 6B demonstrated that mice immunized with HR ⁇ /HBc or HR ⁇ -3 ⁇ /HBc mixture showed about 12% or 10% body weight loss at day 3 post challenge.
- mice received FIRSV intramuscularly showed the highest body weight loss every day and showed about 25% body weight loss at day 3 post challenge.
- the present disclosure provides a better protection to prevent mouse weight loss and an accelerated recovery from initial body weight loss following live RSV challenge. These are evidence that anti-viral immunity elicited by the antigen of the present disclosure confer protection against live RSV A2 strain virus.
- lung samples were fixed in 10% neutral buffered formalin for 24 hrs, embedded in paraffin blocks, sectioned into a thickness of 5 m, and stained with hematoxylin and eosin (H&E).
- H&E hematoxylin and eosin
- lung histopathological changes were observed in the naive group or mice immunized with HBc, HR ⁇ 24, HR ⁇ , HR ⁇ -3 ⁇ or FIRSV, wherein FIRSV immunized mice showed a severe level of histopathology.
- mice received HR ⁇ 24/HBc, HR ⁇ /HBc or HR ⁇ -30/HBc mixture showed none to moderate level of histopathology upon RSV challenge.
- Control of lung viral loads is an important parameter in assessing vaccine efficacy since there would be a positive correlation between viral replication and clinical disease during natural or experimental infections (DeVincenzo, J. P., et al., 2005; Karron, R. A., et al., 1997). Therefore, qRT-PCR targeting RSV N gene was performed to quantify mRNA levels in lung tissues.
- RNA samples were prepared as homogenates using frosted glass slides.
- Total RNA was prepared by Qiagen RNeasy kit from homogenated samples. Two steps qRT-PCR was performed. The first-strand cDNA was amplified from 2 ⁇ g total RNA by SuperScript III Reverse Transcriptase (Invitrogen). The following primer pair was used for qRT-PCR: RSV-A-N-F730: GCAGGATTGTTTATGAATGCC (SEQ ID NO: 35) and RSV-A-N-R857: TCCACAACTTGTTCCATTTC (SEQ ID NO: 36) for RSV subgroup A viruses.
- reactions contained: each primer at 200 nM, 1 ⁇ Power SYBR Green PCR Master Mix (ABI), 1 ⁇ L of cDNA, and water to 10 ⁇ L.
- Real-time PCR was performed using the ABI instrument with the following conditions: 95° C. for 10 min (1 ⁇ ), 95° C. for 15 sec, 60° C. for 60 sec (40 ⁇ ). DNA standards were used to verify the performance of each PCR run and to facilitate the quantification of experimental samples.
- the antigen of the present disclosure can induce both systemic and mucosal antibody responses specific for RSV.
- Mice immunized with the antigen of the present disclosure showed protection against RSV without causing lung disease.
- the antigen of the present disclosure did not over-stimulate lymphocytes compared to FIRSV in a mouse model and offer as a potential safe RSV vaccine candidate.
- sequence of the antigen of the present disclosure is relatively shorter than the wild type RSV F protein and is therefore relatively easy in mass production.
- the antigen of the present disclosure retains only critical antigenic sites and is therefore more helpful in increasing the specificity of antibody identification and avoiding unnecessary reactions such as allergy than wild-type RSV F proteins.
Abstract
Description
TABLE 1 |
Primer sequences |
Primer | Sequence |
HRN-NcoI- |
5′-CCG CCA TGG CCG TGT CTA AGG TGC TGC-3′ |
(SEQ ID NO. 15) | |
HRC-XhoI- |
5′-CAT GCT CGA GCT TGC CGG CGT TCA CAT TG-3′ |
(SEQ ID NO. 16) | |
HRN-A1- |
5′-CAT CGT GAA CAA GCA GAG CGG TTC TGG |
TTC TAA CAG CGA GCT GCT GAG-3′ | |
(SEQ ID NO. 17) | |
HRN-A1- |
5′-CTC AGC AGC TCG CTG TTA GAA CCA GAA |
CCG CTC TGC TTG TTC ACG ATG-3′ | |
(SEQ ID NO. 18) | |
A1-A2- |
5′-GCA GAT CGT GCG GCA GGG TGG TGG TTC |
TTG CAC CGC CAG CAA C-3′ | |
(SEQ ID NO. 19) | |
A1-A2- |
5′-GTT GCT GGC GGT GCA AGA ACC ACC ACC |
CTG CCG CAC GAT CTG C-3′ | |
(SEQ ID NO. 20) | |
A2-HRC- |
5′-AGA CCT TCA GCA ACG GCG GTG GTT CTG |
GTA ACT TCT ACG ACC CCC TGG-3′ | |
(SEQ ID NO. 21) | |
A2-HRC- |
5′-CCA GGG GGT CGT AGA AGT TAC CAG AAC |
CAC CGC CGT TGC TGA AGG TCT-3′ | |
(SEQ ID NO. 22) | |
Site Ø- |
5′-GCC GGA TCC AGC AAC ATC AAG GAG AAC |
AAG TGC AAC GCC GCC AAG AAC TAC ATC GAC | |
AA-3′ | |
(SEQ ID NO. 23) | |
Site Ø- |
5′-GCC AAG CTT CTT GTT CAC GAT GGG CAG |
CAG CTG CTT GTC GAT GTA GTT CTT GGC GGC | |
GTT-3′ | |
(SEQ ID NO. 24) | |
HRN-BamHI- |
5′-GCC GGA TCC AGA ACC AGA ACC GCT CTG |
CTT G-3′ | |
(SEQ ID NO. 25) | |
HRC-EcoRI- |
5′-CGG AAT TCG GTG GTT CTG GTA ACT TCT |
ACG AC-3′ | |
(SEQ ID NO. 26) | |
Site Ø-NheI- |
5′-CTA GCT AGC AGC AAC ATC AAG GAG AAC-3′ |
(SEQ ID NO. 27) | |
Site Ø-BamHI- |
5′-GCC GGA TCC GCC TCC CTT GTT CAC GAT |
GGG CAG C-3′ | |
(SEQ ID NO. 28) | |
Site Ø-BamHI- |
5′-GCC GGA TCC AGC AAC ATC AAG GAG AAC-3′ |
(SEQ ID NO. 29) | |
Site Ø-EcoRI- |
5′-CGG AAT TCG CCT CCC TTG TTC ACG ATG |
GGC AGC-3′ | |
(SEQ ID NO. 30) | |
Site Ø-EcoRI- |
5′-CGG AAT TCA GCA ACA TCA AGG AGA AC-3′ |
(SEQ ID NO. 31) | |
Site Ø-HindIII- |
5′-CCC AAG CTT CTT GTT CAC GAT GGG CAG C-3′ |
(SEQ ID NO. 32) | |
HBc148-NcoI- |
5′-CCG CCA TGG ACA TTG ACC CTT ATA AAG-3′ |
(SEQ ID NO. 33) | |
HBc148-XhoI- |
5′-CAT GCT CGA GAA CAG TAG TTT CCG GAA |
GTG-3′ | |
(SEQ ID NO. 34) | |
Claims (19)
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PCT/CN2017/112361 WO2018095330A1 (en) | 2016-11-22 | 2017-11-22 | Reconstituted rsv antigen |
US16/462,757 US11464850B2 (en) | 2016-11-22 | 2017-11-22 | Recombinant RSV antigens, nucleic acid molecules encoding the antigens, and vaccine compositions comprising the same |
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